, Volume 236, Issue 4, pp 1081–1092

Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response

  • Duygu Kuzuoglu-Ozturk
  • Ozge Cebeci Yalcinkaya
  • Bala Ani Akpinar
  • Geraldine Mitou
  • Gozde Korkmaz
  • Devrim Gozuacik
  • Hikmet Budak
Original Article


An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker® red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8∆::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response.


Abiotic stress Drought tolerance Malondialdehyde Triticum Virus-induced gene silencing Yeast complementation 



Aminopeptidase I


Barley stripe mosaic virus


Coding sequence


Cytoplasm to vacuole targeting


Days post-inoculation


Enhanced chemiluminescence


GAL4 activation domain


Mature API




Open reading frame


Phytoene desaturase




Polyethylene glycol


Precursor API


Quantitative real-time PCR


Reverse-transcriptase PCR


Soil plant analysis development


Virus-induced gene silencing


  1. Bassham DC (2007) Plant autophagy—more than a starvation response. Curr Opin Plant Biol 10:587–593PubMedCrossRefGoogle Scholar
  2. Bassham DC (2009) Function and regulation of macroautophagy in plants. Biochim Biophys Acta Mol Cell Res 9:1397–1403CrossRefGoogle Scholar
  3. Bray EA, Bailey-Serres J, Weretilnyk E (2000) Responses to abiotic stresses. In: Buchanan BB, Gruissem W, Jones RL (eds) Biochemistry and molecular biology of plants. American Society of Plant Physiologists, Rockville, MD, USA, pp 1158–1249Google Scholar
  4. Budak H, Kasap Z, Shearman RC, Dweikat I, Sezerman U, Mahmood A (2006a) Molecular characterization of cDNA encoding resistance gene-like sequences in Buchloe dactyloides. Mol Biotechnol 34:293–301PubMedCrossRefGoogle Scholar
  5. Budak H, Su S, Ergen N (2006b) Revealing constitutively expressed resistance genes in Agrostis species using PCR-based motif-directed RNA fingerprinting. Genet Res Camb 88:165–175CrossRefGoogle Scholar
  6. Cebeci O, Budak H (2009) Global expression patterns of three Festuca species exposed to different doses of glyphosate using the Affymetrix GeneChip Wheat Genome Array. Comp Funct Genomics 2009:505701CrossRefGoogle Scholar
  7. Chung T, Suttangkakul A, Vierstra RD (2009) The ATG autophagic conjugation system in maize: ATG transcripts and abundance of the ATG8-lipid adduct are regulated by development and nutrient availability. Plant Physiol 149:1220–1234Google Scholar
  8. Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra RD (2002) The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. J Biol Chem 277:33105–33114PubMedCrossRefGoogle Scholar
  9. Durmaz E, Coruh C, Dinler G, Grusak MA, Peleg Z, Saranga Y, Yazici A, OzturkL Cakmak I, Budak H (2010) Expression and cellular localization of ZIP1 transporter under zinc deficiency in wild emmer wheat. Plant Mol Biol Rep 29:582–596CrossRefGoogle Scholar
  10. Dvorak J, Akhunov E (2005) Tempos of gene locus deletions and duplications and their relationship to recombination rate during diploid and polyploid evolution in the Aegilops–Triticum alliance. Genetics 171:323–332PubMedCrossRefGoogle Scholar
  11. Ergen ZN, Budak H (2009) Sequencing over 13,000 ESTs from six subtractive cDNA libraries of wild and modern wheats following slow drought stress. Plant Cell Environ 32:220–236PubMedCrossRefGoogle Scholar
  12. Ergen ZN, Dinler G, Shearman RC, Budak H (2007) Identifying, cloning and structural analysis of differentially expressed genes upon Puccinia infection of Festuca rubra var. rubra. Gene 393:145–152PubMedCrossRefGoogle Scholar
  13. Ergen ZN, Thimmapuram J, Bohnert H, Budak H (2009) Transcriptome pathways unique to dehydration tolerant relatives of modern wheat. Funct Integr Genomics 9:377–396PubMedCrossRefGoogle Scholar
  14. Fujiki Y, Yoshimoto K, Ohsumi Y (2007) An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination. Plant Physiol 143:1132–1139PubMedCrossRefGoogle Scholar
  15. Gietz RD, Woods RA (2002) Transformation of yeast by the Liac/SS carrier DNA/PEG method. Methods Enzymol 350:87–96PubMedCrossRefGoogle Scholar
  16. Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata D, Tabata S, Ohsumi Y (2002) Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol 129:1181–1193PubMedCrossRefGoogle Scholar
  17. Harding TM, Morano KA, Scott SV, Klionsky DJ (1995) Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway. J Cell Biol 131:591–602PubMedCrossRefGoogle Scholar
  18. He H, Dang Y, Dai F, Guo Z, Wu J, She X, Pei Y, Chen Y, Ling W, Wu C, Zhao S, Liu JO, Yu L (2003) Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B. J Biol Chem 278:29278–29287PubMedCrossRefGoogle Scholar
  19. Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604–611CrossRefGoogle Scholar
  20. Holzberg S, Brosio P, Gross C, Pogue GP (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J 30:315–327PubMedCrossRefGoogle Scholar
  21. Inoue Y, Suzuki T, Hattori M, Yoshimoto K, Ohsumi Y, Moriyasu Y (2006) AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells. Plant Cell Physiol 47:1641–1652PubMedCrossRefGoogle Scholar
  22. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728PubMedCrossRefGoogle Scholar
  23. Kantar M, Unver T, Budak H (2010a) Regulation of barley miRNA upon dehydration stress correlated with target gene expression. Funct Integr Genomics 10:493–507PubMedCrossRefGoogle Scholar
  24. Kantar M, Lucas S, Budak H (2010b) miRNA expression patterns of Triticum dicoccoides in response to shock drought stress. Planta 233:471–484PubMedCrossRefGoogle Scholar
  25. Ketelaar T, Voss C, Dimmock SA, Thumm M, Hussey PJ (2004) Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins. FEBS Lett 567:302–306PubMedCrossRefGoogle Scholar
  26. Kirisako T, Ichimura Y, Okada H, Kabeya Y, Mizushima N, Yoshimori T, Ohsumi M, Takao T, Noda T, Ohsumi Y (2000) The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol 151:263–276PubMedCrossRefGoogle Scholar
  27. Klionsky DJ, Ohsumi Y (1999) Vacuolar import of proteins and organelles from the cytoplasm. Annu Rev Cell Dev Biol 15:1–32PubMedCrossRefGoogle Scholar
  28. Klionsky DJ, Cregg JM, Dunn WA Jr, Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y (2003) A unified nomenclature for yeast autophagy-related genes. Dev Cell 5:539–545PubMedCrossRefGoogle Scholar
  29. Liu Y, Schiff M, Czymmek K, Talloczy Z, Levine B, Dinesh-Kumar SP (2005) Autophagy regulates programmed cell death during the plant innate immune response. Cell 121:567–577PubMedCrossRefGoogle Scholar
  30. Liu Y, Xiong Y, Bassham DC (2009) Autophagy is required for tolerance of drought and salt stress in plants. Autophagy 5:954–963PubMedCrossRefGoogle Scholar
  31. Mitou G, Budak H, Gozuacik D (2009) Techniques to study autophagy in plants. Int J Plant Genomics 2009:451357PubMedGoogle Scholar
  32. Moriyasu Y, Hattori M, Jauh G, Rogers JC (2003) Alpha tonoplast intrinsic protein is specifically associated with vacuole membrane involved in an autophagic process. Plant Cell Physiol 44:795–802PubMedCrossRefGoogle Scholar
  33. Noda T, Suzuki K, Ohsumi Y (2002) Yeast autophagosomes: de novo formation of a membrane structure. Trends Cell Biol 12:231–235PubMedCrossRefGoogle Scholar
  34. Pogue GP, Lindbo JA, Dawson WO, Turpen TH (1998) Tobamovirus transient expression vectors: tools for plant biology and high-level expression of foreign proteins in plants. In: Gelvin SB, Schilperoot RA (eds) Plant molecular biology manual. Kluwer Academic Publishers, Dordrecht, pp 1–27Google Scholar
  35. Qin G, Ma Z, Zhang L, Xing S, Hou X, Deng J, Liu J, Chen Z, Qu LJ, Gu H (2007) Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development. Cell Res 17:249–263PubMedGoogle Scholar
  36. Simon P (2003) Q Gene: processing quantitative real-time RT-PCR data. Bioinformatics 19:1439–1440PubMedCrossRefGoogle Scholar
  37. Slavikova S, Shy G, Yao YL, Giozman R, Levanony H, Pietrokovski S, Elazar Z, Galili G (2005) The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants. J Exp Bot 56:2839–2849PubMedCrossRefGoogle Scholar
  38. Surpin M, Zheng H, Morita MT, Saito C, Avila E, Blakeslee JJ, Bandyopadhyay A, Kovaleva V, Carter D, Murphy A et al (2003) The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways. Plant Cell 15:2885–2899PubMedCrossRefGoogle Scholar
  39. Thompson AR, Vierstra RD (2005) Autophagic recycling: lessons from yeast help define the process in plants. Curr Opin Plant Biol 8:165–173PubMedCrossRefGoogle Scholar
  40. Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD (2005) Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol 138:2097–2110PubMedCrossRefGoogle Scholar
  41. Thumm M, Egner R, Koch B, Schlumpberger M, Straub M, Veenhuis M, Wolf DH (1994) Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Lett 349:275–280PubMedCrossRefGoogle Scholar
  42. Tsukada M, Ohsumi Y (1993) Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett 333:169–174PubMedCrossRefGoogle Scholar
  43. Unver T, Budak H (2009a) Virus-induced gene silencing, a post transcriptional gene silencing method. Int J Plant Genomics 2009:198680PubMedGoogle Scholar
  44. Unver T, Budak H (2009b) Conserved microRNAs and their targets in model grass species Brachypodium distachyon. Planta 230:659–669PubMedCrossRefGoogle Scholar
  45. Unver T, Bakar M, Shearman RC, Budak H (2010) Genome-wide profiling and analysis of Festuca arundinacea miRNAs and transcriptomes in response to foliar glyphosate application. Mol Genet Genomics 283:397–413PubMedCrossRefGoogle Scholar
  46. Wang CW, Klionsky DJ (2003) The molecular mechanism of autophagy. Mol Med 9:65–76PubMedGoogle Scholar
  47. Wei S, Ma H, Liu C, Wu J, Yang J (2006) Identification and characterization of two rice autophagy associated genes, OsAtg8 and OsAtg4. Mol Biol Rep 33:273–278CrossRefGoogle Scholar
  48. Xiong Y, Contento AL, Bassham DC (2005) AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J 42:535–546PubMedCrossRefGoogle Scholar
  49. Xiong Y, Contento AL, Bassham DC (2007a) Disruption of autophagy results in constitutive oxidative stress in Arabidopsis. Autophagy 3:257–258PubMedGoogle Scholar
  50. Xiong Y, Contento AL, Nguyen PQ, Bassham DC (2007b) Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis. Plant Physiol 143:291–299PubMedCrossRefGoogle Scholar
  51. Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y (2004) Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell 16:2967–2983PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Duygu Kuzuoglu-Ozturk
    • 1
  • Ozge Cebeci Yalcinkaya
    • 1
  • Bala Ani Akpinar
    • 1
  • Geraldine Mitou
    • 1
  • Gozde Korkmaz
    • 1
  • Devrim Gozuacik
    • 1
  • Hikmet Budak
    • 1
  1. 1.Biological Sciences and Bioengineering ProgramSabanci UniversityTuzlaTurkey

Personalised recommendations